Choroidal neovascularization (CNV) represents the most common cause of severe vision loss in patients with age-related macular degeneration (AMD). It is also the most common cause of legal blindness in the United States in patients over the age of 60. Therefore, the study of the diagnosis, prevention and treatment of this condition would have a major impact on the public health. We continue to study the pathogenesis of CNV in a established rodent model of CNV developed in our laboratory. This model uses vascular endothelial growth factor (VEGF) stimulation to induce neovascularization. For the past year, novel findings from this animal model have included: 1. Confirmation that bone marrow derived cells incorporate into the CNV and become part of the endothelium - blood vessel structure. Transplantation of bone marrow cells from a male C57B16 mouse transgenic for the endothelial specific promoter Tie2 driving the LacZ marker gene was performed. These cells were delivered into a female nude mouse previously lethally irradiated. Following 2 months of bone marrow expansion, CNV was induced by our adenovirus VEGF system. By both beta-galactosidase staining and Y-chromosome in-situ, cells derived from the bone marrow transplantation were detected in the CNV within the vascular lumens. 2. Further verification of the extracellular matrix and cellular changes within the choroid was also achieved with findings of macrophage infiltration and perlecan upregulation in the tissue surrounding the CNV. In both cases, these findings were noted before the onset of CNV development. 3. A biopsy specimen of a CNV was examined from a patient with rapidly developing CNV. Immunohistochemistry demonstrated prominent cellular changes within the sub-RPE space. Macrophage staining was intensely positive. Surprisingly, endothelial markers, while positive, were more intense for the immature cell marker CD34 than for vWF. In addition, the majority of endothelial cells were noted to be individual cells rather than within a vascular structure. These findings suggested that a cellular invasion of endothelial cells, potentially immature cells from the bone marrow, were in part responsible for the CNV in this patient. These findings confirmed the oberservations in the animal model. To further verify the ability of bone marrow derived cells to differentiate into endothelial cells, we have modified the standard CAM assay to study the behavior of bone marrow cells. Labeled bone marrow cells are injected into the vasculature of the CAM after application of various growth factors to induce neovascularization. We have found that bone-marrow cells derived from mice will incorporate into the neovascular complexes but only under conditions of G-CSF stimulation of the donor bone marrow and stimulation of the CAM with both pro-inflammatory and angiogenic proteins. Studies are underway to further verify these findings.